EXPRESSION OF THE INFLAMMATORY REGULATOR A20 CORRELATES WITH LUNG FUNCTION IN PATIENTS WITH CYSTIC FIBROSIS

Abstract: Background: A20 and TAX1BP1 interact to negatively regulate NF-
-driven inflammation. A20 expression is altered in F508del/F508del
patients. Here we explore the effect of CFTR and CFTR genotype on A20 and
TAX1BP1expression. The relationship with lung function is also assessed.
Methods: Primary Nasal Epithelial cells (NECs) from CF patients
(F508del/F508del, n=8, R117H/F508del, n=6) and Controls (age-matched,
n=8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene
expression was determined by qPCR.
Results: Silencing of CFTR reduced basal A20 expression. Following LPS
stimulation A20 and TAX1BP1 expression was induced in control NECs and
reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del
had lower expression than R117H/F508del. A20, but not TAX1BP1 expression,
was proportional to FEV1 in all CF patients (r=0.968, p<0.001).
Conclusions: A20 expression is reduced in CF and is proportional to FEV1.
Pending confirmation in a larger study, A20 may prove a novel predictor
of CF inflammation/disease severity.

The renin-angiotensin system and antihypertensive drugs in Alzheimer's disease:current standing of the angiotensin hypothesis?

There is an urgent need to improve upon Alzheimer's disease (AD) treatments. Limitations of existing drugs are that they target specific downstream neurochemical abnormalities while the upstream underlying pathology continues unchecked. Preferable treatments would be those that can target a number of the broad range of molecular and cellular abnormalities that occur in AD such as amyloid-ß (Aß) and hyperphosphorylated tau-mediated damage, inflammation, and mitochondrial dysfunction, as well more systemic abnormalities such as brain atrophy, impaired cerebral blood flow (CBF), and cerebrovascular disease. Recent pre-clinical, epidemiological, and a limited number of clinical investigations have shown that prevention of the signaling of the multifunctional and potent vasoconstrictor angiotensin II (Ang II) may offer broad benefits in AD. In addition to helping to ameliorate co-morbid hypertension, these drugs also likely improve diminished CBF which is common in AD and can contribute to focal Aß pathology. These drugs, angiotensin converting enzyme (ACE) inhibitors, or angiotensin receptor antagonists (ARAs) may also help deteriorating cognitive function by preventing Ang II-mediated inhibition of acetylcholine release as well as interrupt the upregulation of deleterious inflammatory pathways that are widely recognized in AD. Given the current urgency to find better treatments for AD and the relatively immediate availability of drugs that are already widely prescribed for the treatment of hypertension, one of the largest modifiable risk factors for AD, this article reviews current knowledge as to the eligibility of ACE-inhibitors and ARAs for consideration in future clinical trials in AD.

The role of secretory leucoprotease inhibitor in the resolution of inflammatory responses

Chronic lung disease is one of the most common causes of death and disability worldwide. This group of diseases is characterized by a protease burden, an infective process and a dominant pro-inflammatory profile. While SLPI (secretory leucoprotease inhibitor) was initially identified as a serine protease inhibitor, it has since been shown that SLPI possesses other properties distinct from those associated with its antiprotease capabilities that play an important role in protecting the host from infection and injury. In the course of this review, we will highlight the findings from a range of studies that illustrate the multiple functions of SLPI and its role in the resolution of the immune response.

Risk factors, DNA damage, and disease progression in Barrett's esophagus

Esophageal adenocarcinoma develops on a background of Barrett's esophagus. A number of risk factors have been linked to both conditions, including gastroesophageal reflux and smoking. However, the molecular mechanisms by which these factors influence disease progression remain unclear. One possibility is that risk factors generate promutagenic DNA damage in the esophagus. The comet assay was used to measure DNA damage in esophageal (Barrett's and squamous) and gastric mucosa of Barrett's patients with (n = 24) or without (n = 50) associated adenocarcinoma or high-grade dysplasia in comparison with control patients (squamous mucosa) without Barrett's esophagus (n = 64). Patients completed a questionnaire detailing exposure to some of the known risk factors for Barrett's esophagus and adenocarcinoma. In Barrett's esophagus patients, DNA damage was higher in Barrett's mucosa compared with normal esophageal and gastric mucosa (P < 0.001). In addition, the highest quartile of DNA damage in Barrett's mucosa was associated with an increased risk (odds ratio, 9.4; 95% confidence interval, 1.1-83.4; P = 0.044) of developing adenocarcinoma or high-grade dysplasia compared with DNA damage levels in the lowest quartile. Smoking was associated with higher DNA damage in squamous epithelium in all patient groups (P < 0.01) and in Barrett's mucosa (P < 0.05) in Barrett's esophagus patients only. In controls only, current reflux was associated with higher DNA damage, whereas anti-inflammatory drug use resulted in lower levels. Collectively, these data imply a genotoxic insult to the premalignaint Barrett's mucosa that may explain the genetic instability in this tissue and the progression to adenocarcinoma. There is an indication for a role for smoking in inducing DNA damage in esophageal mucosa but an understanding of the role of reflux requires further investigation.

Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis.

Organizational Attributes of Practices Successful at a Disease Management Program

Objective: To assess the contribution of organizational factors to implementation of 3 asthma quality measures: enrollment in a disease management program, development of a written treatment plan, and prescription of severity-appropriate anti-inflammatory therapy. Study design: A total of 138 pediatric clinicians and 247 office staff in 13 urban clinics and 23 nonurban private practices completed questionnaires about their practice's organizational characteristics (eg, leadership, communication, perceived effectiveness, job satisfaction). Results: 94% of the clinicians and 92% of the office staff completed questionnaires. When adjusted for confounders, greater practice activity and perceived effectiveness in meeting family needs were associated with higher rates of enrollment in the Easy Breathing program, whereas higher scores for 3 organizational characteristics-communication timeliness, decision authority, and job satisfaction-were associated with both higher enrollment and a greater number of written treatment plans. None of the organizational characteristics was associated with greater use of anti-inflammatory therapy. Conclusions: Three organizational characteristics predicted 2 quality asthma measures: use of a disease management program and creation of a written asthma treatment plan. If these organizational characteristics were amenable to change, then our findings could help focus interventions in areas of effective and acceptable organizational change. © 2009 Mosby, Inc. All rights reserved.

Angiogenic potential of vitreous from proliferative diabetic retinopathy and eales' disease patients

Proliferative Diabetic Retinopathy (PDR) and Eales' Disease (ED) have different aetiologies although they share certain common clinical symptoms including pre-retinal neovascularization. Since there is a need to understand if the shared end-stage angiogenic pathology of PDR and ED is driven by common stimulating factors, we have studied the cytokines contained in vitreous from both patient groups and analyzed the angiogenic potential of these samples in vitro.

Vitreous samples from patients with PDR (n = 13) and ED (n = 5) were quantified for various cytokines using a cytokine biochip array and sandwich ELISA. An additional group of patients (n = 5) with macular hole (MH) was also studied for comparison. To determine the angiogenic potential of these vitreous samples, they were analyzed for their ability to induce tubulogenesis in human microvascular endothelial cells. Further, the effect of anti-VEGF (Ranibizumab) and anti-IL-6 antibodies were studied on vitreous-mediated vascular tube formation.

Elevated levels of IL-6, IL-8, MCP-1 and VEGF were observed in vitreous of both PDR and ED when compared to MH. PDR and ED vitreous induced greater levels of endothelial cell tube formation compared to controls without vitreous (P<0.05). When VEGF in vitreous was neutralized by clinically-relevant concentrations of Ranibizumab, tube length was reduced significantly in 5 of 6 PDR and 3 of 5 ED samples. Moreover, when treated with IL-6 neutralizing antibody, apparent reduction (71.4%) was observed in PDR vitreous samples.

We have demonstrated that vitreous specimens from PDR and ED patients share common elevations of pro-inflammatory and pro-angiogenic cytokines. This suggests that common cytokine profiles link these two conditions.

Antibiotic and anti-inflammatory therapies for cystic fibrosis

Cystic fibrosis (CF) lung disease is characterized by chronic bacterial infection and an unremitting inflammatory response, which are responsible for most of CF morbidity and mortality. The median expected survival has increased from <6 mo in 1940 to >38 yr now. This dramatic improvement, although not great enough, is due to the development of therapies directed at secondary disease pathologies, especially antibiotics. The importance of developing treatments directed against the vigorous inflammatory response was realized in the 1990s. New therapies directed toward the basic defect are now visible on the horizon. However, the impact of these drugs on downstream pathological consequences is unknown. It is likely that antibiotics and anti-inflammatory drugs will remain an important part of the maintenance regimen for CF in the foreseeable future. Current and future antibiotic and anti-inflammatory therapies for CF are reviewed. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

The role of antimicrobial peptides in periodontal disease.

Objectives Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

The role of the cathelicidin LL-37 in periodontal disease.

Abstract Background Fibroblasts respond to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. As such fibroblasts play a significant role as regulators of the host response in periodontal disease. LL-37, an antimicrobial peptide, found in saliva and GCF, inhibits LPS-induced cytokine signalling in macrophages, suggesting a role in host defence in periodontal disease. This study investigated the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and also its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (0.01µg/ml) along with LL-37 (0-50µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Microarray analysis revealed that LL-37 upregulated a significant number of cytokines and chemokines by > 5 fold. The stimulatory effect on IL-8 mRNA expression was confirmed by Q-PCR. Conclusion LL-37 appears to have pleiotrophic effects in innate immunity. Its ability, at low concentrations, to reduce bacterial LPS-induced cytokine production in gingival fibroblasts suggests a potential therapeutic role in the management of periodontal disease.

Targeting the complement system for the management of retinal inflammatory and degenerative diseases

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states.

Nonsteroidal Anti-Inflammatory Drug Use is Not Associated With Reduced Risk of Barrett’s Esophagus

OBJECTIVES: Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced risk of esophageal adenocarcinoma. Epidemiological studies examining the association between NSAID use and the risk of the precursor lesion, Barrett’s esophagus, have been inconclusive.

METHODS: We analyzed pooled individual-level participant data from six case-control studies of Barrett’s esophagus in the Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON). We compared medication use from 1474 patients with Barrett’s esophagus separately with two control groups: 2256 population-based controls and 2018 gastroesophageal reflux disease (GERD) controls. Study-specific odds ratios (OR) and 95% confidence intervals (CI) were estimated using multivariable logistic regression models and were combined using a random effects meta-analytic model.

RESULTS: Regular (at least once weekly) use of any NSAIDs was not associated with the risk of Barrett’s esophagus (vs. population-based controls, adjusted OR = 1.00, 95% CI = 0.76–1.32; I2=61%; vs. GERD controls, adjusted OR = 0.99, 95% CI = 0.82–1.19; I2=19%). Similar null findings were observed among individuals who took aspirin or non-aspirin NSAIDs. We also found no association with highest levels of frequency (at least daily use) and duration (≥5 years) of NSAID use. There was evidence of moderate between-study heterogeneity; however, associations with NSAID use remained non-significant in “leave-one-out” sensitivity analyses.

CONCLUSIONS: Use of NSAIDs was not associated with the risk of Barrett’s esophagus. The previously reported inverse association between NSAID use and esophageal adenocarcinoma may be through reducing the risk of neoplastic progression in patients with Barrett’s esophagus.

Pharmacogenetics and nutrigenetics in crohn’s disease

Tese de doutoramento, Biologia (Genética), Universidade de Lisboa, Faculdade de Ciências, 2015

Development of CO-releasing molecular for the treatment of inflammatory diseases

Dissertation presented to obtain a Ph.D. degree (Doutoramento) in Chemistry at the Instituto de Tecnologia Quimica e Biol6gica da Universidade Nova de Lisboa